The 5’-AMP-Activated Protein Kinase (AMPK) Is Involved in the Augmentation of Antioxidant Defenses in Cryopreserved Chicken Sperm

Abstract

Semen cryopreservation is a unique tool for the management of animal genetic diversity. However, the freeze-thaw process causes biochemical and physical alterations which make difficult the restoration of sperm energy-dependent functions needed for fertilization. 5’-AMP activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy metabolism. Mitochondria functions are known to be severely affected during sperm cryopreservation with deleterious oxidative and peroxidative effects leading to cell integrity and functions damages. The aim of this study was thus to examine the role of AMPK on the peroxidation/antioxidant enzymes defense system in frozen-thawed sperm and its consequences on sperm functions. Chicken semen was diluted in media supplemented with or without AMPK activators (AICAR or Metformin [MET]) or inhibitor (Compound C [CC]) and then cryopreserved. AMPKα phosphorylation, antioxidant enzymes activities, mitochondrial potential, ATP, citrate, viability, acrosome reaction ability (AR) and various motility parameters were negatively affected by the freeze-thaw process while reactive oxygen species (ROS) production, lipid peroxidation (LPO) and lactate concentration were dramatically increased. AICAR partially restored superoxide dismutase (SOD), Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), increased ATP, citrate, and lactate concentration and subsequently decreased the ROS and LPO (malondialdehyde) in frozen-thawed semen. Motility parameters were increased (i.e., + 23% for motility, + 34% for rapid sperm) as well as AR (+ 100%). MET had similar effects as AICAR except that catalase activity was restored and that ATP and mitochondrial potential were further decreased. CC showed effects opposite to AICAR on SOD, ROS, LPO and AR and motility parameters. Taken together, our results strongly suggest that, upon freeze-thaw process, AMPK stimulated intracellular anti-oxidative defense enzymes through ATP regulation, thus reducing ROS and lipid peroxidation, and consequently partially restoring several essential sperm functions and leading to a better quality of cryopreserved sperm.

Effect of metformin on semen quality

“…Moreover, metformin was found to enhance the phosphorylation of 5'-AMP activated protein kinase (AMPK), which is a sensor protein of cellular energy status, thus, a strategic enzyme in regulating energy balances (i.e., glucose and lipid metabolism) (Bertoldo et al, 2015;Hardie et al, 2003;Ma et al, 2015;Nguyen et al, 2014Nguyen et al, , 2015. Therefore, metformin may improve sperm function via enhancing the activity of AMPK.…”

“…Similar findings of enhancing the antioxidant function were observed when using diet-induced obesity rats (i.e., higher superoxide dismutase and glutathione peroxidase, and lower malondialdehyde) (Fang et al, 2012). In fact, metformin was identified as having antioxidant activity against oxidative damage by reactive oxygen species (Cahova et al., 2015;Ouslimani et al, 2005).Moreover, metformin was found to enhance the phosphorylation of 5'-AMP activated protein kinase (AMPK), which is a sensor protein of cellular energy status, thus, a strategic enzyme in regulating energy balances (i.e., glucose and lipid metabolism) (Bertoldo et al, 2015;Hardie et al, 2003;Ma et al, 2015;Nguyen et al, 2014Nguyen et al, , 2015. Therefore, metformin may improve sperm function via enhancing the activity of AMPK.…”

L’AMPK, régulateur de l’énergie et des fonctions des spermatozoïdes

“…restauration de la spermatogenèse, et une augmentation de la concentration des spermatozoïdes et de leur motilité [39,40]. Au cours des dernières années, l'implication de l'AMPK dans la régula-tion des concentrations de ROS ou de LPO, ainsi que dans l'activité des principales enzymes antioxydantes présentes au niveau des spermatozoïdes, a été mise en évidence [41]. Chez le coq, l'ajout, avant cryoconservation de sperme, d'activateur de l'AMPK (metformine ou AICAR [acadesine]), permet ainsi d'augmenter la phosphorylation de la kinase après décongélation tout en diminuant la production de LPO et de ROS.…”

“…‡ [21]. Chez le coq, une production d'ATP et de lactate a pu être observée dans les spermatozoïdes après stimulation par des activateurs pharmacologiques de l'AMPK (metformine ou AICAR) [4,41]. L'AICAR stimule également la production de citrate, un intermédiaire du cycle de Krebs.…”

“…Dans ces conditions particulières, les spermatozoïdes nécessitent une quantité très importante d'énergie pour restaurer leur pouvoir fécondant. L'ajout de metformine ou d'AICAR avant cryoconservation permettrait donc de restaurer une partie de cette énergie nécessaire, en stimulant le métabolisme anaérobie et, pour l'AI-CAR, le métabolisme aérobie [41]. L'AMPK est donc essentielle pour maintenir, dans les spermatozoïdes, un niveau d'énergie qui est nécessaire pour la conduite de la fécondation, en particulier en conditions « stressantes ».…”